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Long reads RNA-seq Alignment

Learn how to perform read alignment and prepare BAM files for use with ScanMST.

Learning Objectives

By the end of this tutorial, you will be able to:

  • Necesary files preparation
  • Alignment file (BAM) files generation

Prerequisites:

  • SAMtools installed
  • Minimap2 installed
  • Basic command-line experience

Time: Approximately 30 minutes to several hours, depending on the size of the FASTQ file.

Necessary files preparation

# Download reference genome FASTA
wget https://hgdownload.soe.ucsc.edu/goldenPath/hg38/bigZips/hg38.fa.gz
gunzip hg38.fa.gz

# Download reference annotation GTF
wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_48/gencode.v48.annotation.gtf.gz
gunzip gencode.v48.annotation.gtf.gz

# Convert GTF/GFF to BED12
paftools.js gff2bed gencode.v48.annotation.gtf > annotation.bed12

PacBio Iso-Seq Data

minimap2 -t 16 -R "@RG\tID:sample\tSM:hs\tLB:ga\tPL:PACBIO" -Y --cs -ax splice:hq -uf --secondary=no --junc-bed annotation.bed12 hg38.fa FLNC_reads.fastq | samtools sort -@ 8 -O BAM -o sample.bam - && samtools index sample.bam sample.bai

ONT direct RNA Data

minimap2 -t 16 -R "@RG\tID:sample\tSM:hs\tLB:ga\tPL:NANOPORE" -Y --cs -ax splice -uf -k14 --junc-bed annotation.bed12 hg38.fa directRNA.fastq | samtools sort -@ 8 -O BAM -o sample.bam - && samtools index sample.bam sample.bai

ONT direct cDNA Data

The raw FASTQ file from Nanopore caller should not used, you have to prepossess with Pychopper

minimap2 -t 16 -R "@RG\tID:sample\tSM:hs\tLB:ga\tPL:NANOPORE" -Y --cs -ax splice -uf -k14 --junc-bed annotation.bed12 hg38.fa directcDNA_pychopper.fastq | samtools sort -@ 8 -O BAM -o sample.bam - && samtools index sample.bam sample.bai

Expected Output

  1. BAM: BAM file with SA and cs tags.

Check if the BAM file with SA and cs tags

samtools view isoseq_data.bam|grep "SA:Z"|less

samtools view isoseq_data.bam|grep "cs:Z"|less

Summary

You've learned how to:

  • ✅ Long read RNA-seq alignment for ScanMST.

Long read alignment files are Ready!

The alignment files (BAM format) are now ready for MST calling with ScanMST!